Proteoglycans (PGs) play an important role in a variety of diseases effecting the excretory system, respiratory system, circulatory system, skeletal system, as well as the multisystem diseases of aging 1 and cancer 2. Progress has been made in the isolation, purification, and characterization of PGs from extracellular matrix; these advances have recently led to attempts to sequence PG core-proteins 3. The chemistry and biology of a PG, however, is dominated by the polysaccharide or glycosaminoglycan (GAG) components for which it is named. The characterization of these GAG components has primarily been directed towards the identification of the class to which they belong (ie, chondroitin, dermatan, heparan, etc), their molecular weight, and their charge density (ie, degree of sulfation). A new approach is required to elevate the structural characterization of the PG's GAG portion to the level currently being practiced on the core protein. The specific aims of this proposal are to: 1. Develop general methods for sequencing the polysaccharide component of proteoglycans; 2. Explore the chondroitin/dermatan and heparin/heparin families of proteoglycans for the presence of ordered, defined sequences; and 3. Determine whether there are differences in sequence in a given class of PG's which are derived from different tissue sources. The long term objective of this proposed research is to better understand the physiological role of a major component of the extracellular matrix, proteoglycans, by obtaining an intimate and detailed knowledge of their structure. Unlike nucleic acids and proteins, polysaccharides are polydisperse mixtures and cannot easily be prepared as homogeneous, pure substances. Even if such an approach were possible, the purification of a single homogeneous chain for sequencing raises a serious question. Will the arbitrary choice of a single chain truly reflect the structure and the biological function of the polysaccharide mixture as a whole? We propose to sequence GAG polysaccharides as a mixture. Three approaches will be used to sequence GaGs. These are: 1. sequencing by simulation, using computer and mathematical models; 2. kinetic sequencing of homogeneous GAG derived oligosaccharides; and 3. defined reading frame sequencing. Each approach will use microgram quantities of GAGs prepared from PGs under mild conditions. Following enzymatic depolymerization, analysis will be carried out by gradient PAGE using autoradiographic detection or by SAX-HPLC using radiochemical detection.